14.02 Characterising neuronal subtype vulnerability to HIV-1 infection as ascertained by neurotransmitter expression in a human neuronal model <strong>of</strong> HIV-1 dise Palser A, Benson L, Jauniaux E, Miller D, Weiss R, Trillo-Pazos G University College London, Institute <strong>of</strong> Neurology and Windeyer Institute HIV-1 associated dementia (HAD) is associated with microgliosis, gliosis and neuronal damage. Clinically, it manifests with motor and cognitive impairments 10-25 years after initial HIV-1 infection. HIV-1 infection is correlated with latent and lytic infection in different brain cells. HIV-1 infection <strong>of</strong> neurones has been demonstrated ex-vivo and in-vitro. We have shown that HIV-1 infection <strong>of</strong> primary human neural cultures (70-80% neurones, 10-20% astrocytes, 5% oligodendrocytes, no microglia) results in cellular functional damage at the tripartite synapse similar to that observed in cases with HAD. We know that 10- 20% <strong>of</strong> cells are infected within this neural culture model <strong>of</strong> grey matter. We are currently characterising specific neuronal subtype vulnerability to HIV-1 infection (3 to 21 days post-infection) by immunoblotting and RT-PCR to neurotransmitter synthetic enzymes (dopamine, serotonin, acetylcholine, GABA, glutamate, epinephrine, norepinephrine) and calcium binding proteins in these neural cultures. Expression <strong>of</strong> neurotransmitter synthetic enzymes for dopamine and serotonin are downregulated as a result <strong>of</strong> HIV-1 infection at 3-21 days post-infection. Moreover, infectious virus is produced at these time points within this neural culture system as assessed in chemokine transfected NP2 cells. These results suggest that there is low level release <strong>of</strong> infectious viral particles in culture and that Dopamine and serotonin producing neuronal subtypes are targeted by HIV-1 within neural culture system. These findings suggest that disruption <strong>of</strong> neurotransmitter synthetic pathways might be implicated in neuronal damage in HIV-1 disease in the brain. 14.03 Effects <strong>of</strong> anaesthetics on basal and stimulated dopamine release, measured by microdialysis Young A M J, Dixon A L School <strong>of</strong> Psychology,, University <strong>of</strong> Leicester,, Lancaster Road,, Leicester, LE1 9HN Many experiments investigating brain function, using neurochemical, electrophysiological and imaging techniques, are performed in anaesthetised animals for practical or ethical reasons. However, despite the widespread use <strong>of</strong> anaesthetised animals in these types <strong>of</strong> study, very little is known about the effect <strong>of</strong> anaesthetics on brain function, and in particular on neurotransmitter release. The present study measured the effect <strong>of</strong> anaesthesia on basal and stimulated (potassium, amphetamine or mild footshock) levels <strong>of</strong> dopamine and its metabolites, DOPAC and HVA, in rat nucleus accumbens using brain microdialysis. The anaesthetics employed were halothane, urethane and α-chloralose, all <strong>of</strong> which are commonly used for experiments in animals. Results showed that with halothane, relatively stable baseline levels <strong>of</strong> dopamine and its metabolites were achieved, which were similar to levels measured in unanaesthetised controls. With urethane normal levels <strong>of</strong> dopamine were measured, but metabolite levels were significantly decreased, while under α-chloralose dopamine was significantly raised, and metabolites were lowered compared to controls. Under halothane and urethane, changes in dopamine and its metabolites during stimulation were similar to those seen in unanaesthetised animals, whereas under α- chloralose the effects were markedly attenuated. Thus, mesolimbic dopamine mechanisms remain relatively intact under halothane and urethane anaesthesia. Under α-chloralose, however, both basal and stimulated levels <strong>of</strong> dopamine are severely affected. We suggest, therefore, that halothane is the most appropriate anaesthetic for studying mesolimbic dopamine function in anaesthetised animals, although urethane may be suitable under certain circumstances: α-chloralose, however is not suitable, as the dopamine system is severely compromised under this anaesthetic. 14.04 Effect <strong>of</strong> NMDA receptor activation in the lateral entorhinal area on the left dorsal hippocampal glutama, and aspartate release in two animal models Brady A T, De Souza I E J, McCabe O M, Moran M P, O'Shea S D, O'Connor W T Applied Neurotherapeutics Research Group, UCD School <strong>of</strong> Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland We employed dual probe microdialysis in the lateral entorhinal area (LEA) and dorsal hippocampus (DH) in the 82 day old adult rat (P82) to investigate the effects <strong>of</strong> intra-LEA perfusion with (NMDA 300μM, 20 min) on dialysate glutamate and aspartate levels in the DH <strong>of</strong> socially isolated and maternally deprived rats. Isolated rats were weaned on P25 and housed individually for 55 days while maternally deprived rat pups experienced a single 24-hour period <strong>of</strong> maternal deprivation on P9. Socially reared rats acted as control. Basal dialysate glutamate levels were stable over the duration <strong>of</strong> the experiment. DH glutamate levels (чM) were similar in the social control, isolated and maternally deprived rat (0.263*0.072), (0.192*0.038) and (0.208*0.025) respectively. Aspartate levels (чM) were similar in the social control (0.036*0.004) isolated (0.039*0.008) and maternally deprived (0.059*0.010) groups. Intra-LEA NMDA was associated with a 39%, 20% and 36% increase in DH glutamate release in social control, isolated and maternally deprived rats. In contrast DH aspartate levels were significantly increased in the maternally deprived group (p=0.0.0042 v’s social control). A maximal increase <strong>of</strong> 42% (p=0.0129) and (p=0.0038) in the isolated group and maternally deprived group respectively. Thus DH aspartate appears to be more responsive than glutamate to LEA NMDA receptor activation. It is currently believed that glutamate is the main neurotransmitter carried by the perforant pathway from the LEA to the DH. The findings from the present study suggest that aspartate also plays a role in perforant pathway transmission. 14.05 Novel mutations in the human GlyT2 gene in hyperekplexia Carta E, Chung SK, Remy N, Vanbellinghen JF, Bours V, Sameer M. Zuberi, John B.P. Stephenson, Marina A.J. Tijssen, Arn M.J.M. van den Maagdenberg, Brian R. Pearce, Kirsten Harvey, Stéphane Supplisson, Mark I. Rees, Robert J. Harvey The School <strong>of</strong> Pharmacy, London; Univ. Wales Swansea; Liège University Hospital, Belgium; Royal Hospital for Sick Children, Glasgow; Academic Medical Centre, Amsterdam and Leiden Univ. Medical Centre, The Netherlands; Laboratoire de Neurobiologie Moléculaire et Cellulaire, Paris The glycine transporter GlyT2 has an essential role in maintaining a high presynaptic pool <strong>of</strong> neurotransmitter at glycinergic synapses. We have recently shown that missense, nonsense and frameshift mutations in the GlyT2 gene (SLC6A5) can cause hyperekplexia. Since our initial study, we have discovered further mutations in SLC6A5 in individuals from the UK, Holland, France, Spain and Italy. In some <strong>of</strong> these additional hyperekplexia cases, only one coding mutation has been found. Since most mutations in the GlyT2 gene are inherited as compound heterozygotes (i.e. one mutation on each allele) it is possible that: i) there is a deletion affecting one or more exons in the second allele, which would not be easily detectable by PCR and sequencing; ii) that the single ‘orphan’ mutations exert dominant-negative effects (as previously described for mutation S510R) or iii) that uncharacterised alternative splicing <strong>of</strong> the GlyT2 gene has excluded some exons from genetic screening. To assess the latter possibility, we performed 5’ and 3’ RACE on human spinal cord cDNA. This revealed a number <strong>of</strong> additional splice variants <strong>of</strong> GlyT2 specifying novel N- and C- termini. In particular, a new C-terminal GlyT2 variant (denoted hGlyT2B) lacks the C-terminal class III PDZ binding motif (TQC) found in GlyT2A that is crucial for interactions with the PDZ domain-containing protein syntenin-1. Since syntenin-1 has been proposed to regulate the trafficking and/or presynaptic localization <strong>of</strong> GlyT2, hGlyT2B is likely to be distinct in terms <strong>of</strong> subcellular location and/or interacting partners. Page 26/101 - 10/05/2013 - 11:11:03
15.01 L-serine enhances the inhibition <strong>of</strong> calcium currents by anandamide in cultured sensory neurones from neonatal rats Khairy H, Ross R, Pertwee R, Scott R School <strong>of</strong> Medical Sciences, University <strong>of</strong> Aberdeen, Institute <strong>of</strong> Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK. The endocannabinoid anandamide (AEA) has previously been found to inhibit high voltage-activated Ca2+ currents in cultured dorsal root ganglion (DRG) neurones (Evans et al., 2004 Br. J. Pharmacol. 141: 1223-1233). Using the whole cell recording patch clamp technique with choline chloride-based extracellular solution and CsCl-based patch pipette solution we have investigated the modulation <strong>of</strong> AEA responses by 1 µM L-serine in cultured DRG neurones. High voltage-activated Ca2+ currents were evoked from a holding potential <strong>of</strong> -90 mV by voltage step commands to 0mV. Application <strong>of</strong> 1µM AEA for 3-5 minutes inhibited the mean peak Ca2+ current by 34 ± 12 % (n=7; P
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